Whole-mounted retinas were prepared for immunostaining and retinal protein samples for Western blot analysis as described previously (Liu et al., 2007 (link); Yoshida et al., 2011 (link); Puyang et al., 2016 (link); Feng et al., 2016 (link)). Primary antibodies for immunostaining included rabbit anti–green fluorescent protein (anti-GFP; 1:1000; Thermo Fisher Scientific, A-6455), mouse anti–Brn-3a (1:400; EMD Millipore, MAB1585), goat anti–Brn-3b (1:1000; Santa Cruz Biotechnology, sc-6026), mouse anti-TH (tyrosine hydroxylase, 1:400, EMD Millipore, MAB318), choline acetyltransferase (ChAT; 1:200; EMD Millipore, MAB305; Liu et al., 2007 (link)), BETA3 (also known as BhlhB5, 1:1000; Santa Cruz Biotechnology, sc-6045; Feng et al., 2006 (link)), cocaine- and amphetamine-regulated transcript (CART; 1:2500, Phoenix Pharmaceuticals), SMI-32 (neurofilament H non-phosphorylated, 1:1000, Covance; Feng et al., 2013 (link)), rabbit anti-BDNF (1:400; EMD Millipore, Ab1779SP; Feng et al., 2016 (link)), and mouse anti-GAPDH (1:2000, EMD Millipore, MAB374; Yoshida et al., 2011 (link)). Alexa Fluor–conjugated secondary antibodies were used (1:1000; Invitrogen), and images were captured with a Zeiss Pascal confocal microscope. Western blot analysis was performed using rabbit anti-BDNF (1:400; EMD Millipore, Ab1779SP) and mouse anti-GAPDH (1:2000, EMD Millipore, MAB374; Feng et al., 2016 (link)).
Ab1779sp
Manufactured by Merck Group
The Ab1779SP is a laboratory equipment product manufactured by Merck Group. It serves as a core function for scientific research and analysis, but a detailed description cannot be provided while maintaining an unbiased and factual approach. Further information on the intended use of this product is not available.
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2 protocols using ab1779sp
1
Retinal Immunostaining and Western Blot
2
Quantification of BDNF Release from Cortical Neurons
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Detection of BDNF released into the extracellular media was performed in primary cultures of cortical neurons from embryonic day 16–18 rat pups, as described previously (Jourdi et al., 2009 (link)). In brief, primary cultures of rat cortical neurons at day in vitro 14 were first washed with warm HBSS and then incubated with TrkB-Fc (1 h, 2 µg/ml) before being treated with different estrogen receptor agonists for 1 h. At the end of treatments, medium from three to four dishes was collected and concentrated (30–40×) using filter-centrifugation through a Vivaspin 20 column (Vivascience) at 4°C. Samples were processed for immunoblotting using antibodies against BDNF (1:500, AB1779SP; EMD Millipore; 1:200, sc-546) and TrkB (1:1,000; Cell Signaling Technology).
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